The immune response to tumors arising from a chemically induced adenocarcinoma in inbred rats ('13762') has been characterized. 13762 cells express Tumor Rejection Antigens (TRA) and a T cell-mediated immune response to 13762 cells can be induced by immunization with irradiated cells. Syngeneic Rat1 fibroblasts transformed by expression of activated ras oncogene ('Rat1/ras') can immunize rats to resist tumorigenic challenges of 13762 cells and also induce anti-tumor T cells which crossreact with 13762 cells in Winn assays. The proposed research shall: identify TRA molecules expressed by 13762 adenocarcinoma and also shall investigate the mechanism by which these antigenic tumorigenic cells escape immune response. Two hypotheses will be tested: that TRA expressed by 13762 tumor is derived from activated ras protein, and that expression of the immunomodulatory cytokine Transforming Growth Factor beta-1 by 13762 tumor is responsible for selective immunosuppression of anti-tumor immune response. To pursue the hypothesis that cross-reactive TRA molecules expressed by 13762 (and Rat1/ras) cells are derived from ras protein, in vitro proliferation and IL-2 or IFN release assays will be performed using anti-tumor T cells and synthetic peptides corresponding to selected regions of the ras protein sequence. In addition, the ability to directly immunize rats with synthetic peptides to resist 13762 (or Rat1/ras) tumorigenic challenges will be tested. In addition,TRA molecules shall be purified from 13762 cells. Anti-tumor T cells will be prepared, T-T hybridomas made by in vitro fusion with BW 5147 and tumor reactive T cell hybridomas will be cloned. Anti-tumor T cell hybridomas will form the basis of an in vitro antigen presenting system that shall be used to screen HPLC chromatography fractions derived from 13762 tumor. Purified TRA molecules will be characterized in biochemical terms and identified by deriving a partial amino acid sequence. The hypothesis that 13762 cells express Transforming Growth Factor beta-1 (TGF) which down-regulates anti-tumor immune response will be tested. Cell lines will be made in which TGF expression will be extinguished by means of antisense technology. Using the derived TGF-minus 13762 cells tumorigenicity will be correlated with TGF expression. The mechanism of activation of latent TGF elaborated by tumorigenic cells will be investigated.
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